High-sensitivity magnetic marker used for immune response measurement

ABSTRACT

Disclosed are a highly-sensitive magnetic marker for use in measuring an immunoreaction with a SQUID magnetic sensor and the preparation thereof. The magnetic marker is composed of a magnetic fine particle and a polymer encapsulating the particle, wherein the particle diameter of the magnetic fine particle (preferably ferrite Fe 3 O 4 ) is 20 to 40 nm and the external the diameter of the magnetic marker is 40 to 100 nm, the polymer having carboxyl groups on the surface thereof. The magnetic maker for a SQUID magnetic sensor is prepared by causing the surface of a magnetic fine particle to adsorb a hydrophilic macromonomer (preferably polyvinylpyrrolidone) having a polymerizable vinyl group at the terminal thereof and having a molecular weight of 500 to 1000, and then adding a monomer of a hydrophilic vinyl compound having carboxyl group and a crosslinking agent for carrying out copolymerization reaction.

TECHNICAL FIELD

The present invention belongs to the technical field of immunoreactionmeasurement, and particularly relates to a highly-sensitive magneticmarker for use in measuring an immunoreaction utilizing a SQUID magneticsensor.

BACKGROUND ART

Measurement of an immunoreaction, i.e. an antigen-antibody reaction, iswidely applied in various areas such as in the detection of germs ormicrobes, disease diagnosis, gene analysis, measurement of environmentalsubstances and so on. An immunoreaction determination is done bymeasuring the specific binding of a target substance (antigen) with atest reagent (antibody) so as to qualify and/or quantify the targetsubstance.

Hitherto immunoreaction measurement has been primarily conducted bymeans of the spectroscopic method: A test reagent (antibody) is providedwith a spectroscopic marker such as a fluorescence-labeled enzyme, andan immunoreaction (antigen-antibody) reaction is detected by measuringthe light emitted from the marker. However, while there is increasingdemand for highly-sensitive and rapid detection of a microchemicalreactions, the existing systems do not meet the requirements in manycases. Thus, there is a desire for a new type of immunoreactionmeasurement system with high sensitivity.

Recently, the SQUID (superconducting quantum interference device) hasreceived considerable attention as a highly-sensitive magnetic sensor,as it enables the measurement of a very week magnetic field by takingadvantage of a quantum effect (the quantization of magnetic flux). Themost significant application of the SQUID is the measurement of thebrain magnetic field, in which a magnetic field generated by the brainis measured to analyze or diagnose brain function. Other applicationshave started to emerge in various areas such as medical science,material evaluation, material analysis, precision measurement, naturalresources survey and so on, and it is also proposed to apply the SQUIDto immunoreaction measurement (cf. for example, K. Enpuku“Antigen-antibody reaction measurement utilizing SQUID,” Ouyou-butsuri,Vol. 70, No. 1, p48-49 (2001)).

In an immunoreaction measurement system utilizing the SQUID, an antibodymaterial is attached to the surface of a magnetic marker composed of apolymer encapsulating a fine magnetic material. An antigen-antibodyreaction will take place between the antibody and an antigen (the targetsubstance) to produce a weak magnetic field signal attributable to themagnetic marker, which is measured by a SQUID (see FIG. 1). The generalpractice is to fix the SQUID and move the sample to be measured for thedetection of a magnetic signal.

While the immunoreaction measurement system using the SQUID is ahighly-sensitive sensor ascertained to be about ten times more sensitivethan the fluorescent antibody method (see the reference mentionedabove), further improvement is expected to produce a detection systemfor immunoreactions with still higher sensitivity. One approach to suchimprovement of the immunoreaction detection system using the SQUID is todevelop an optimized magnetic marker while also improvinginstrumentation such by lowering noise (noise reduction).

However, there is found no prior art developed through a systematicstudy of the conditions to be met in improving the sensitivity of amagnetic marker for use in the SQUID magnetic sensor. For example,although reference is made in WO96/27133 (PCT/EP 96/00823) to magneticparticles for immunoassay including a magnetic sensor using the SQUID,there are no concrete disclosures of technologies for improving thesensitivity of a magnetic marker for use in the SQUID magnetic sensor.It is mentioned that the size of the magnetic particles ranges widelyfrom 1 to 1000 nm, but this can be considered to be an arbitrarydefinition not based on technical studies into magnetic particle size.Moreover, no concrete disclosures are found on the type of polymers forobtaining a highly-sensitive magnetic marker or the production thereof.

The magnetically labeled antibody discussed above, in which a magneticparticle is encapsulated within a polymer and an antibody is bound tothe surface of the polymer, has primarily been used to the purificationand separation of antibodies. When commercially available magneticparticles are used for this purpose, the diameter of the magneticparticle is about 10 to 15 nm, while the size of polymer particle (i.e.the external diameter of the assembly as a whole) is 50 to 1000 nm.However, such conventional magnetically labeled antibodies cannot beapplied to detect an antigen-antibody reaction with high sensitivity,because the properties of the magnetic particles are insufficient forsuch applications.

The object of the present invention is to provide a novel technologyrelating to a highly-sensitive magnetic marker suitable for use in themeasurement of an immunoreaction using the SQUID magnetic sensor.

DISCLOSURE OF INVENTION

After extensive studies, the present inventors achieved the presentinvention by noting that the size of the magnetic fine particlecomposing the core of the magnetic marker, and also the size of thepolymer particle encapsulating the magnetic fine particle (morestrictly, the external diameter of the magnetic marker as a whole), areparameters that affect the sensitivity of a magnetic marker for theSQUID magnetic sensor, and they successfully designed a polymersynthesizing system that ensures the preparation of a magnetic marker inwhich these parameters are optimized.

Thus, according to the present invention there is provided a magneticmarker composed of a magnetic fine particle and a polymer encapsulatingthe particle, for use in measuring an immunoreaction with a SQUIDmagnetic sensor, wherein the particle diameter of said magnetic fineparticle is 20 to 40 nm and the diameter (external diameter) of saidmagnetic marker is 40 to 100 nm, said polymer having carboxyl groups onthe surface thereof. In a preferred embodiment of the magnetic marker ofthe present invention, the magnetic fine particle is composed mostly ofFe₃O₄.

The present invention also provides a method for preparing theabove-mentioned magnetic marker for use in a SQUID magnetic sensor,which comprises the steps of (i) causing the surface of a magnetic fineparticle to adsorb a hydrophilic macromonomer having a polymerizablevinyl group at the terminal thereof and having a molecular weight of 500to 1000, and then (ii) adding a monomer of hydrophilic vinyl compoundhaving carboxyl groups and a crosslinking agent for carrying out acopolymerization reaction. In a preferred embodiment of the method forpreparing the magnetic marker for a SQUID magnetic sensor according tothe present invention, the macromonomer for use in the synthesis of thepolymer is polyvinylpyrrolidone, polyoxyethylene or polyacrylamide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically shows the principle of measuring an immunoreactionby a SQUID magnetic sensor using the magnetic marker of the presentinvention.

FIG. 2 illustrates a reaction scheme according to the present invention,in which a magnetic fine particle is encapsulated (coated) with apolymer, as well as the chemical formulae of the reactants used in thereaction.

FIG. 3 shows an example of the adsorption isotherms in the case where amagnetic fine particle is made to adsorb a macromonomer according to thepresent invention.

FIG. 4 shows an example of the particle diameter distribution ofparticles (magnetic markers) obtained by encapsulating (coating)magnetic fine particles with a polymer according to the presentinvention.

FIG. 5 shows an electromicroscopic (SEM) view of an unmodified ferritefine particle prior to the polymer-encapsulation (polymer-coating)according to the present invention.

FIG. 6 shows an electromicroscopic (SEM) view of a composite particle (amagnetic marker) prepared by the polymer-encapsulation (polymer-coating)according to the present invention.

FIG. 7 graphically shows an example of the results obtained when anantibody was adsorbed onto a magnetic marker of the present invention.

FIG. 8 shows an example of the relationship between the weight of themagnetic fine particle contained in the magnetic marker of the presentinvention and the SQUID output.

FIG. 9 shows an example of the results of protein detection experimentsusing an antibody-bound magnetic marker of the present invention,illustrating the relationship between the quantity of the protein andthe SQUID output.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention emerged from step-by-step studies carried out todetermine the dominant factors affecting the sensitivity of a magneticmarker for use in a SQUID magnetic sensor and culminated in theattainment of an extremely highly-sensitive magnetic marker. Theembodiments of the present invention will be detailed below withreference to such factors.

(1) Magnetic Fine Particle and its Size:

The present inventors discovered that the size (the diameter) of amagnetic fine particle encapsulated by a polymer to be applied as amagnetic marker for use in a SQUID magnetic sensor should be larger thanthat of the commercially available magnetic fine particle mentionedpreviously; that is to say, the diameter is required to be 20 to 40 nm.This is because the magnetic signal from the magnetic fine particle isproportional to the volume of the fine particle, and hence a largerparticle will produce a larger magnetic signal. The increase in thevolume of the magnetic fine particle will also induce a change in themagnetic characteristics: A small particle will exhibit so-calledsuperparamagnetism whereas a large particle will exhibit residualmagnetism. This also contributes to the enhancement of the magneticsignal.

The minimal diameter for a particle to develop magnetism as mentionedabove is supported by a theoretical calculation as follows: When thevolume of a magnetic fine particle is represented by V and the magneticanisotropy energy thereof is represented by K, the transition fromsuperparamagnetism to residual magnetism takes place at the pointdefined by the equation KV/k_(B)T=20, where k_(B) is Boltzmann constantand T is 300K. In the case of using Fe₃O₄ as the magnetic fine particle,it is estimated that K=10 to 20 (kJ/m³). This corresponds to a diameterof the fine particle of d=20 to 25 nm. It can thus be seen that the sizeof the magnetic fine particle is desirably d>20 nm.

It is crucial for a magnetically labeled antibody, to which the presentinvention is directed, to possess a sufficient dispersibility, since theantibody is used so as to bind with an antigen (a target substance to bemeasured) in an aqueous medium. Poor dispersibility will inhibit theantigen-antibody reaction. If the size of the magnetic fine particle istoo large, significant sedimentation of the particles will occur alongwith poor dispersibility. In order to avoid these issues, it isnecessary for the specific gravity of the polymer for encapsulating themagnetic fine particle (more strictly, the specific gravity of themagnetic marker) as a whole to be kept at approximately 1 to 3. Thisalso means that the size of the magnetic fine particle is required to bed<40 nm.

While any of various materials can be used as the magnetic fineparticle, including magnetite, Fe₂O₃ and Fe₃O₄, ferrite Fe₃O₄ is mostpreferably used since it exhibits the maximal magnetism.

(2) External Diameter of Magnetic Marker:

In a magnetic marker for a SQUID magnetic sensor of the presentinvention, it is also essential for the diameter of the polymer particle(more strictly, the external diameter of the magnetic marker as a whole)to be 40 nm or larger and 100 nm or smaller. This is because, if thepolymer size is too large in the detection of an immunoreaction (anantigen-antibody binding reaction), the binding between the magneticallylabeled antibody and the antigen will not proceed efficiently. Amagnetic marker having too-large particle size (external diameter) isalso undesirable because of poor dispensability that readily causessedimentation, as mentioned above with reference to the magnetic fineparticle.

(3) Polymer to be Used:

The magnetic marker for a SQUID magnetic sensor having theabove-mentioned characteristics can be optimally prepared by takingadvantage of the polymer system designed by the present inventors.Specifically, according to the present invention, the encapsulation orcoating of a magnetic fine particle with a polymer can be effectivelyconducted by causing the surface of the magnetic fine particle to adsorba hydrophilic macromonomer having a polymerizable vinyl group at theterminal thereof and having a molecular weight of 500 to 1000, and thenadding a monomer of hydrophilic vinyl compound having carboxyl groupsand a crosslinking agent for carrying out a copolymerization reaction,thereby producing the magnetic marker for a SQUID sensor, wherein theparticle diameter of the magnetic fine particle is 20 to 40 nm and theexternal diameter of the magnetic marker is 40 to 100 nm, the polymerhaving carboxyl groups on the surface thereof.

While a particularly preferred example of the macromonomer for use ispolyvinylpyrrolidone, other polymers such as polyoxyethylene orpolyacrylamide can be employed. The adsorption of such a macromonomeronto the magnetic fine particle is generally carried out by dispersingmagnetic fine particles, typified by ferrite Fe₃O₄, in methanol, andadding the macromonomer into the dispersion, followed by stirring forseveral hours at room temperature.

The magnetic fine particles with the macromonomer adsorbed thereon arethen dispersed in a low-polar solvent (e.g. tetrahydrofuran), so thatthe surface of the magnetic polymer particle is encapsulated or coatedwith the polymer through copolymerization (radical polymerization) ofthe crosslinking agent and the monomer. A trivinyl compound is generallyused as the crosslinking agent. As the monomer, a vinyl compound ispreferably used which possesses carboxyl groups and is a hydrophilicmolecule as a whole. The use of a hydrophobic monomer with a long alkylchain having no hydrophilic carboxyl groups will result in a magneticmarker exhibiting poor dispersibility.

Thus, the polymer system employed in the present invention is based on aquite novel technical idea of encapsulating or coating a magnetic fineparticle. Regarding a magnetic material utilizing polyvinylpyrrolidone,a process is known in which a magnetic powdery material is admixed witha vinylpyrrolidone-vinyl acetate copolymer resin (Japanese PatentApplication Publication No. 2000-28616). However, it is apparent thatthe method of the present invention is totally different from suchprocess.

According to the present invention, it is possible to encapsulate amagnetic fine particle, such as of ferrite Fe₃O₄, homogeneously with thesynthetic polymer to a uniform thickness, and what is more, theresultant magnetic fine particle-synthetic polymer composite is impartedwith a desired amount of carboxyl groups on the surface thereof.Specifically, the polymer may have on the surface thereof 500 to 5000,desirably 2000 to 3000, carboxyl groups per particle of the magneticmarker.

According to the present invention, it is also possible to control theparticle diameter of the magnetic marker so as to be in the range of 40to 100 nm by adjusting the conditions in the respective steps of themethod of the present invention, i.e., the steps of causing the surfaceof a magnetic fine particle to adsorb a macromonomer and then adding amonomer having carboxyl groups to enable reaction with a crosslinkingagent for radical copolymerization. In addition, the method of thepresent invention makes it possible to encapsulate magnetic fineparticles with the monomer having carboxyl groups individually withoutinducing aggregation among the p articles.

(4) Characteristic Properties of the Magnetic Marker:

The magnetic marker for a SQUID magnetic sensor of the present inventionas prepared in the above-mentioned manner has excellent dispersibility,and the dispersion in an aqueous medium is stably retained generally fora period of longer than one month.

The magnetic marker of the present invention for a SQUID sensorpossesses a number of carboxyl groups on the surface thereof, and henceis capable of binding antibodies thereon via the carboxyl groups. Theantibody binding ability of the magnetic marker of the present inventionis very high: For example, it was found to be capable of binding arabbit IgG with a yield of 80% or more.

The magnetic marker of the present invention, when bound with anantibody, is subject to the process of measuring the immunoreaction(antigen-antibody reaction) as explained previously. The sensitivity inthe measurement is extremely high: For example, it is possible tomeasure an antigen (a protein) down to even as little as 1 pg (picogram)or less.

EXAMPLE

The present invention will be explained more specifically with referenceto the following working examples, which are not for restricting thepresent invention.

Example 1 Preparation of Ferrite Fine Particle Encapsulated with Polymer

In accordance with the reaction schemes shown in FIG. 1, there wereprepared polymer-encapsulated ferrite fine particles having carboxylgroups on the surface thereof (magnetic marker for a SQUID magneticsensor).

<Adsorption of Polyvinylpyrrolidone onto Ferrite Fine Particle>

Into methanol 10 ml there was dissolved polyvinylpyrrolidone (molecularweight: 520), as the macromonomer, in an amount in the range of 0.004 to0.04 g, followed by addition of fine particles of ferrite Fe₃O₄ 0.05 g(Toda-Kogyo Ltd., particle diameter: 25 nm) and then the resultant wassubjected to ultrasonic irradiation. Following gentle stirring for fourhours, the particles having polyvinylpyrrolidone adsorbed thereon wereisolated with a centrifuge and then subjected to drying in vacuo.

The amount adsorbed was calculated from the loss in weight during theprocess over a temperature rise from 100 to 800° C. FIG. 3 shows theadsorption isotherm. It was found that the amount ofpolyvinylpyrrolidone adsorbed leveled off when it reaches 1.0×10⁻³ molper 1 gram of the ferrite particle.

<Polymer Encapsulation of Ferrite Fine Particle via RadicalCopolymerization>

The amount adsorbed was calculated from the loss in weight during theprocess over a temperature rise from 100 to 800° C. FIG. 3 shows theadsorption isotherm. It was found that the amount ofpolyvinylpyrrolidone adsorbed leveled off when it reaches 1.0×10⁻³ molper 1 gram of the ferrite particle.

<Polymer Encapsulation of Ferrite Fine Particle via RadicalCopolymerization>

The ferrite fine particles having the hydrophilic macromonomer adsorbedthereon were dispersed in tetrahydrofuran, for polymer encapsulation orcoating through the copolymerization between the crosslinking agent(trivinyl compound) and the monomer in the presence of AIBN(2,2′-azobis(isobutyronitrile)) (polymerization initiator), as detailedbelow.

<Encapsulation 1: Polymer-Encapsulation Around Ferrite Fine ParticleThrough the Copolymerization Between Tri(1-acryloyloxyethyl)amineHydrochloride (a) and N-acryloyl-1-aminopentane (b)>

Into tetrahydrofuran 5 ml, there were dissolved N-acroylaminopentanate0.12 g and 0 to 100 times in amount of tri(1-acryloyloxyethyl)aminehydrochloride, (tri (acroyloxy) ethylene) amine hydrochloride, as thecrosslinking agent, in which the quantity of N-acroylaminopentanate was100 times that of vinyl groups of the polyvinylpyrrolidone adsorbed ontothe ferrite fine particle. To the resultant solution there were addedferrite fine particles 0.018 g and 2,2′-azobis(isobutyronitrile) 0.01 g,in which the ferrite particle had polyvinylpyrrolidone adsorbed thereonat the rate of polyvinylpyrrolidone 0.2 g per one gram of the ferriteparticles. The solution was stirred at 65° C. for ten hours. Thecomposite particles were separated from the solution with a centrifuge.The procedures were repeated five times, followed by separation of theunreacted monomer and crosslinking agent.

Table 1 shows the amounts of the polymer on the surfaces of the ferritefine particles thus prepared. As shown in Table 1 the amount of thepolymer bound increased with increasing amount of the crosslinkingagent, whereas the fact that no aggregation occurred among the particlescan be seen from the fact that the particle diameter was of the order of29 to 30 nm as measured by the dynamic light scattering method (DLS). Itshould be noted that particle size measured by DLS is generally smallerthan the actual size observed by a microscope, as will be explainedlater. The composite particles prepared in the above manner exhibited arelatively short period of retaining the dispersion, i.e. two days atthe maximum. This is probably because the long methylene chain, which ishydrophobic, induces some degree of aggregation among the particles dueto the low polar interaction in the aqueous medium. TABLE 1 Crosslinking Amount of Particle agent (a) Monomer (b) polymer size Dispersion10⁻³ mol/g- 10⁻³ mol/g- bound diameter retaining Entry Fe₃O₄ Fe₃O₄mg/g-Fe₃O₄ nm period 1 0 37.7 527 32 two days 2 0.4 37.7 656 30 Sixhours 3 3.7 37.7 706 30 Six hours 4 18.9 37.7 733 30 Six hours 5 37.737.7 866 29 Six hours<Encapsulation 2: Polymer-Encapsulation Around Ferrite Fine ParticleThrough Copolymerization Between Tri(1-acryloyloxyethyl)amineHydrochloride (a) and N-acryloylglycine (c)>

This encapsulation was carried out in the same manner as inEncapsulation 1 above. The results are given in Table 2. The amount ofthe polymer bound increased with increasing amount of the crosslinkingagent, with a maximum value of approx. 870 mg/g. Of the compositeparticles, the particles having the polymer bound in an amount of 650 to700 mg/g exhibited a particularly stable dispersion. with the dispersionin the aqueous medium being retained for longer than four weeks. Theamount of the carboxyl groups on the surface also increased withincreasing amount of the crosslinking agent, with a maximum value of 60μmol/g. The particle diameter was measured by the DLS method. TABLE 2Cross-kinking Amount of Amount of agent (a) Monomer (c) polymer Particlecarboxyl Dispersion 10⁻³ mol/g- 10⁻³ mol/g- bound diameter groupsretention Entry Fe₃O₄ Fe₃O₄ mg/g-Fe₃O₄ nm μmol/g period 1 0 39.8 527 3426.6(1050) 4 weeks 2 0.4 39.8 656 26 28.6(1200) Longer than 4 weeks 34.0 39.8 706 29 41.4(1800) Longer than 4 weeks 4 19.9 39.8 733 2551.9(2350) 2 weeks 5 39.8 39.8 866 33 59.7(2900) 2 weeksThe numerical values in ( ) indicate the number of carboxyl groupscalculated as being present on the polymer surface per particle.<Encapsulation 3: Polymer-Encapsulation Around Ferrite Fine ParticleThrough Copolymerization Between Tri(1-acryloyloxyethyl)amineHydrochloride (a) and N-acryloylglutamic Acid (d)>

The encapsulation was carried out in the same manner as inEncapsulation 1. The results are given in Table 3. The particle diameteras shown in the table was measured by the DLS method. In thisencapsulation it was also found that there occurred no aggregation amongthe particles and that the amount of the polymer bound increased withincreasing amount of the crosslinking agent, with a maximum value of 947mg/g. FIG. 4 shows the particle-diameter distribution (measured by theDLS method) of the composite particle given as Entry 4 in Table 4. Itcan be seen that there were no particles of a large diameter due to theaggregation. The amount of the carboxyl groups on the surface alsoincreased with increasing the amount of the crosslinking agent, with amaximum value of 97 mmol/g. This value corresponds to 0.7 carboxylgroups per square nanometer of the surface of the particle. All thecomposite particles prepared by this encapsulation produced a stabledispersion in the aqueous medium, which was retained for a period oflonger than four weeks. TABLE 3 Cross-linking Amount of Amount of agent(a) Monomer (d) polymer Particle carboxyl Dispersion 10⁻³mol/g-10⁻³mol/g- bound diameter groups retention Entry Fe₃O₄ Fe₃O₄ mg/g-Fe₃O₄nm μmol/g period 1 0 31.7 433 34 37.8(1400) Longer than 2 0.3 31.7 47630 47.5(1750) 4 weeks 3 3.2 31.7 648 26 73.6(3100) 4 15.8 31.7 871 2792.7(4500) 5 31.7 31.7 947 27 97.2(4850)The numerical values in ( ) indicate the number of carboxyl groupscalculated as being present on the polymer surface per particle.

FIG. 5 and FIG. 6 are microscopic (SEM) views of unmodified ferrite fineparticles and polymer-encapsulated (Encapsulation 3) ferrite fineparticles, respectively. It is seen from the SEM view of the unmodifiedferrite fine particles that aggregation was produced among the particlesduring the drying process in the preparation of the sample, whereas itwas confirmed that the polymer-encapsulated particles developedexcellent dispersion with the diameter (the external diameter) of theparticle being about 80 nm.

The amount of carboxyl groups on the polymer surface as shown in Table 2and Table 3 was determined as follows: To dehydrated, distilledchloroform 5 ml, there were added composite particles(polymer-encapsulated ferrite fine particles) 10 mg andN,N′-dicyclohexylcarbodiimide 15 mg, followed by stirring for two hoursat room temperature. To the resultant dispersion was added p-nitrophenol15 mg, followed by stirring for twelve hours at room temperature.Following the separation of unreacted p-nitrophenol from the compositeparticles using a centrifuge, the particles were subjected to drying invacuo. Then, the particles having the p-nitrophenolate groups thereonwere weighed and dispersed in a 4% ammonia aqueous solution, followed bygentle stirring for twelve hours. The solution in which p-nitrophenolwas liberated was isolated from the composite particles by centrifuging,and then the solutions were combined, giving a total volume of 10.0 ml.The amount of p-nitrophenol contained in the aqueous solution thusobtained was determined through the absorbance at 400 nm (molarabsorptivity ε=18000).

Example 2 Binding of Antibody

Polymer-encapsulated ferrite particles 0.017 g (magnetic marker),prepared in the manner of Encapsulation 3 of Example 1, were dispersedin a phosphate buffer solution (pH 7.8) 5 ml, followed by the additionof 1-ethyl-3-(3-dimethylaminopropyl) carbodiimido hydrochloride 0.01 g.Following stirring of the resultant solution for one hour at 4° C.,there was added 0.016 mg of rabbit antibody (9.3 mg per gram of the fineparticles) and then the solution was stirred for six hours at roomtemperature. The particles having the antibody bound thereon wereseparated from the phosphate buffer solution using a centrifuge. Theamount of the antibody bound was 7.0 mg/g. The amount of the antibodybound was calculated from the amount of the antibody fed minus theamount of the nonbound antibody, wherein the amounts were determinedthrough the absorbance at 280 nm.

FIG. 7 shows the results of the binding of the IgG onto thepolymer-encapsulated ferrite fine particles. In the case where about 10mg of the antibody was added per gram of the particles, it is indicatedthat approx. 80% of the antibody was successfully bound onto theparticles. It is thus evidenced that the composite particle (themagnetic marker) prepared according to the present invention exhibits ahigh ability of binding an antibody thereon.

Example 3 Relation Between Magnetic Material and SQUID Output

The magnetic signal from a magnetic marker of the present invention wasmeasured by a SQUID magnetic sensor; the magnetic marker was composed ofFe₃O₄ fine particles with a diameter of 25 nm, as prepared byEncapsulation 3 of Example 1, and the polymer encapsulating the particleand having carboxyl groups on the surface thereof, and had an externaldiameter of 80 nm. FIG. 8 shows the results of the SQUID outputmeasurements against varying weight of the magnetic marker. The ordinateis the weight of the ferrite fine particle (pg) in the magnetic markerwhile the abscissa is the SQUID output (mΦ₀). As can be seen from thefigure, there is a good relationship between the weight of the markerand the SQUID output. As it is possible for a SQUID sensor to make ameasurement to a level of 0.1 mΦ₀ or lower, the figure indicates thatthe magnetic marker of the present invention enables the measurement ofthe ferrite magnetic fine particle down to even an amount of less than 1pg.

Example 4 Relation Between Antibody-Bound Magnetic Marker and SQUIDOutput

Detection of an antigen (protein) was carried out using the magneticmarker having the antibody bound thereon as prepared in Example 2,together with the SQUID magnetic sensor. Thus, the protein was specificto the rabbit IgG, and the amount of the protein which was bound to theantibody was determined through the magnetic signal from the magneticmarker. FIG. 9 shows the results of the SQUID output measurementsagainst the amount of the protein. The abscissa is the weight of theprotein (pg), while the ordinate is the SQUID output (mΦ₀). As can beseen from the figure, there is a good relationship between the weight ofthe protein and the SQUID output. As it is possible for a SQUID sensorto make a measurement to a level of 0.1 mΦ₀ or lower, the figureindicates that the magnetic marker enables the measurement of theprotein down to even an amount as low as about 0.2 pg.

INDUSTRIAL UTILITY

As can be seen from the above explanation, the magnetic marker of thepresent invention enables the measurement of an immunoreaction(antigen-antibody) reaction with extremely high-sensitivity, andtherefore is expected to make a large contribution in a number offields, including medical fields current achieving rapid progress, bymaking it possible to measure biological substances which have beenconventionally impossible to measure.

1. A magnetic marker composed of a magnetic fine particle and a polymerencapsulating the particle, for use in measuring an immunoreaction witha SQUID magnetic sensor, wherein the particle diameter of said magneticfine particle is 20 to 40 nm and the external diameter of said magneticmarker is 40 to 100 nm, said polymer having carboxyl groups on thesurface thereof.
 2. The magnetic marker as claimed in claim 1, whereinthe magnetic fine particle is composed of ferrite Fe₃O₄.
 3. The magneticmarker as claimed in claim 2, wherein the polymer has, on the surfacethereof, 500 to 5000 carboxyl groups per particle of the magneticmarker.
 4. The magnetic marker as claimed in claim 3, wherein thepolymer has, on the surface thereof, 2000 to 3000 carboxyl groups perparticle of the magnetic marker.
 5. A method for preparing a magneticmarker composed of a magnetic fine particle and a polymer encapsulatingthe particle, for use in measuring an immunoreaction with a SQUIDmagnetic sensor, wherein the particle diameter of the magnetic fineparticle is 20 to 40 nm and the external diameter of the magnetic markeris 40 to 100 nm, said polymer having carboxyl groups on the surfacethereof, which method comprises the steps of (i) causing the surface ofa magnetic fine particle to adsorb a hydrophilic macromonomer having apolymerizable vinyl group at the terminal thereof and having a molecularweight of 500 to 1000, and then (ii) adding a monomer of a hydrophilicvinyl compound having carboxyl groups and a crosslinking agent forcarrying out copolymerization reaction.
 6. The method for preparing themagnetic marker as claimed in claim 5, wherein the macromonomer ispolyvinylpyrrolidone, polyoxyethylene or polyacrylamide.